The long term objective of this research proposal is to gain new insights[unreadable] into the molecular and cellular events that are caused by encountering[unreadable] foreign antigens and generate the appropriate immune responses. The[unreadable] ligation of receptors on T cells with their counter receptors on APCs[unreadable] initiates cell-mediated immune responses. The molecular mechanisms by[unreadable] which multiple extracellularly engaged receptors regulated intracellular[unreadable] biochemical response is of prime importance in modern immunology. We have[unreadable] recently developed a highly sensitive and quantitative 3-dimensional (3-[unreadable] D) immunofluorescence workstation to study at the single cell level[unreadable] physiological immune interactions. Our previous studies at the single[unreadable] cell level showed that engaged receptors and their associated proteins[unreadable] cluster at the T-APC contact area. Early 3-D studies indicate that[unreadable] multiple adhesion and signaling molecules cluster ind distinct domains[unreadable] at the cell-cell contacts. In the present proposal we will use the new[unreadable] imaging system to study at the single cell level the detailed spatial and[unreadable] temporal molecular rearrangements that occur at the T-APC contacts during[unreadable] physiological interactions. We will then attempt to determine the[unreadable] mechanisms that are responsible for these regulated rearrangements and[unreadable] their functional significance for the relevant immune responses. The[unreadable] specific aims are:[unreadable] Aim 1. To study the temporal and 3-dimensional spatial redistribution of[unreadable] T cell receptors (TCR, CD4, FLA-1, CD28, CD45) and their colocalization[unreadable] with intracellular signaling and regulatory proteins that are implicated[unreadable] in regulating T cell responses (talin, PKCtheta, P-Tyr, grb2, lck, fyn,[unreadable] zap-70, PI-3K, PLCgamma1, and ras-GAP) in Ag-induced T-APC conjugates in[unreadable] order to determine their functional involvement in physiological immune[unreadable] interactions.[unreadable] Aim 2. To determine the role of CD28 and to identify its associated[unreadable] proteins during physiological Th-APC by repeating the experiments in Aim[unreadable] 1 with a cloned Th cell that does not express CD28 but retained[unreadable] expression of TCR, CD4, LFA-1 and CD45. To confirm causality by re-[unreadable] expressing either full length CD28 or a truncated CD28 that lacks its[unreadable] entire cytoplasmic domain.[unreadable] Aim 3. To determine by structure-function analysis the structural basis[unreadable] for the selective clustering of PKCtheta, but not any of the other[unreadable] expressed PKCs, at the cell contract along with CD28. To identify signals[unreadable] that cause this unique PKC translocation with the aid of well-defined[unreadable] PDGF-receptor signaling mutants.[unreadable] It is expected that these novel studies at the single cell level will[unreadable] significantly increase our understanding of the molecular events that[unreadable] occur early in the immune response and determine its outcome. This[unreadable] knowledge may be useful in the long term in enhancing immune surveillance[unreadable] and in improving the design of new vaccines.[unreadable]